Non-therapeutic methods for alleviating or reducing stress symptoms of ruminants

ABSTRACT

The present invention relates to the reduction of stress in ruminants in need thereof.

This application is the U.S. national phase of International ApplicationNo. PCT/EP2019/051595 filed Jan. 23, 2019 which designated the U.S. andclaims priority to EP Patent Application No. 18153285.4 filed Jan. 24,2018, the entire contents of each of which are hereby incorporated byreference.

The present invention relates to the reduction of stress in ruminants inneed thereof.

Methylsulfonylmethane (MSM, also known as dimethylsulfone), is anaturally occurring organosulfur compound and a putative methyl donorwhich is known to have several health benefits such as in particularnormalizing the body functions in animals displaying symptoms of stress.

Methylsulfonylmethane naturally occurs in body fluids and tissues aswell as in a variety of fresh foods including most fruits andvegetables, milk, and grains. Furthermore, it is marketed as a dietarysupplement for humans and animals. Although present in a variety offoods, methylsulfonylmethane is readily lost due to its volatile nature.In addition, methylsulfonylmethane is not yet everywhere approved asfeed additive. Thus, there is an ongoing need for ingredients whichstimulate the endogenous production of methylsulfonylmethane in order toprofit from its extraordinary health benefits.

Surprisingly it has now been found, that the supplementation of themethane inhibitors propanediol mononitrate respectively chloroform toruminants leads to a significant increase in methylsulfonylmethane inthe rumen fluid. Furthermore, animals in stress situations, when treatedwith propanediol mononitrate beforehand, show less symptoms of stresscompared to respective control groups.

Thus, the present invention relates to a methane inhibitor respectivelya composition comprising a methane inhibitor for the treatment orprophylaxis of the symptoms of stress in ruminants.

In a particular embodiment the invention relates to a method for thenon-therapeutic alleviation or prophylaxis of the symptoms of stress inruminants, said method including administering a methane inhibitor or acomposition comprising a methane inhibitor to a ruminant prior toexperiencing stress, while experiencing stress and/or after havingexperienced stress.

In another embodiment, the present invention relates to a method forenhancing the endurance to stress and/or lessening the symptoms ofstress in ruminants, said method encompassing the step of administeringan effective amount of a methane inhibitor to a ruminant in needthereof. Preferably, the methane inhibitor is administered in the formof a composition comprising the respective methane inhibitor, such as inthe form of a premix, a feed composition or a feed additive.

In another embodiment, the invention relates to the use of a methaneinhibitor for increasing endogeneous methyl donors such as in particularmethylsulfonylmethane in the rumen fluid of a ruminant, in particular toenhance the endurance to stress and/or lessen the symptoms of stress.

The term methane inhibitor relates to all compounds suitable to reducethe methane emissions in ruminants (i.e. rumen methane inhibitors).Suitable methane inhibitors according to the present invention includegarlic extracts, allicin, propanediol mononitrate, chloroform, nitrate,nitroethane, lauric acid, lauricidin as well as marine algae such as theHawaiian micro-algae Chaetoceros without being limited thereto.Preferred methane inhibitors in all embodiments of the present inventionare garlic extracts, allicin, chloroform and propanediol mononitrate.Most preferred in all embodiments of the present invention is themethane inhibitor propanediol mononitrate, which is particularlyeffective.

The term ‘stress’ as used herein is defined as the symptom resultingfrom exposure to a situation or environment that is not normal for ananimal. Stress can often be seen when animals are handled and/ortransported.

It is well understood by a person skilled in the art that the treatmentof stress is non-therapeutically, e.g. to prevent or minimizedeterioration of the well-being or health of a ruminant in periods ofstress such as e.g. heat stress and/or stress due to transportationand/or slaughtering.

Advantageously, the methane inhibitor respectively the compositioncomprising the methane inhibitor is administered to the ruminant priorto the ruminant experiencing stress, while the ruminant is experiencingstress and/or after the ruminant experiences stress.

Thus, in a particular embodiment, the present invention relates to amethod for the non-therapeutic alleviation or prophylaxis of thesymptoms of stress in ruminants, said method including administering amethane inhibitor or a composition comprising a methane inhibitor to aruminant prior to experiencing stress, while experiencing stress and/orafter having experienced stress said method encompassing the consecutivesteps of a.) assessing a present or future stress situation for aruminant (in order to determine a ruminant in need of treatment) and b.)administering to the ruminant in need thereof an effective amount of themethane inhibitor.

In an advantageous embodiment, the present invention relates to thetreatment (alleviation) of or prophylaxis against the symptoms of stressinduced by temperature (heat), poor ventilation, overcrowding,transportation, slaughtering as well as pests, preferably induced byheat, transportation and/or slaughtering.

The symptoms of stress in ruminants include lethargy, decrease in orlack of appetite, an elevated or increased respiratory rate, signs ofdehydration and/or an elevated heart rate.

Thus, in another embodiment the present invention relates to a methaneinhibitor or a composition comprising the methane inhibitor as well asany methods and uses thereof according to the present invention for thetreatment (alleviation) of or prophylaxis against lethargy, a decreasein or lack of appetite, an elevated or increased respiratory rate,dehydration and/or an elevated heart rate.

In one advantageous embodiment the present invention is directed tomethods for feeding a methane inhibitor respectively a compositioncomprising a methane inhibitor to ruminants experiencing heat stress orthat may become heat stressed, for example, due to high temperatures,humidity, dew points or combinations thereof.

In another advantageous embodiment, the present invention is directed tomethods for preventing or minimizing deterioration of the well-being orhealth of a ruminant during heat stress, said method includingdetermining a ruminant is experiencing heat stress or may become heatstressed followed by feeding said ruminant an effective amount of amethane inhibitor respectively a composition comprising a methaneinhibitor.

Periods of heat stress can easily be determined by a person skilled inthe art e.g. by observing two or more of an about 10 percent decrease indry matter intake, reduced performance, increased respirations, elevatedinternal body temperature, open mouth breathing, increased panting,sweating, or failed reproduction. Alternatively or in addition thepotential for heat stress can be easily be determined based on one ormore of historical weather patterns or short-term weather forecasts.

In a further advantageous embodiment, the present invention relates to amethod for preventing or minimizing deterioration of the well-being orhealth of a ruminant during transportation and/or slaughteringcomprising the step of administering said ruminant with an effectiveamount of a methane inhibitor respectively a composition comprising amethane inhibitor for an effective period of time.

In the present context, a ruminant is a mammal of the order Artiodactylathat digests plant-based food by initially softening it within theanimal's first stomach, known as the rumen, then regurgitating thesemi-digested mass, now known as cud, and chewing it again. The processof again chewing the cud to further break down plant matter andstimulate digestion is called “ruminating”.

Ruminants according to the present invention include cattle, goats,sheep, giraffes, American bison, European bison, yaks, water buffalo,deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, andnilgai.

Propanediol mononitrate (also referred to 1,3-propanediol mononitrate orPDMN [CAS-No: 100502-66-7]) is a known compound which can e.g. bemanufactured as outlined in WO2004043898 or WO2012084629 and isavailable at DSM Nutritional Products Ltd.

The methane inhibitor, respectively the composition according to thepresent invention are preferably administered to the ruminant for aneffective period of time sufficient to enhance the endurance to stressand/or lessen the symptoms of stress.

In all embodiments of the present invention the methane inhibitor suchas in particular the propanediol mononitrate or chloroform can beadministered as such or in the form of a composition comprising saidmethane inhibitor.

In any or all of the embodiments herein, the methane inhibitorrespectively the composition comprising the methane inhibitor ispreferably administered to the animal for an effective period of time toenhance the endurance to stress and/or lessen the symptoms of stress,particularly at set intervals. The set interval can be daily, or it canbe more or less frequently than that. Preferably, the set interval isdaily.

The effective period of time may be easily selected by a person skilledin the art based on the symptoms and the severity of the symptoms or theenvisaged stress situation, which is to come and may be at least 1 day,at least 3 days, at least 7 days, at least 30 days, at least 60 days, orat least 90 days or a period of time lasting from 1 day to 200 days,from 1 day to 90 days, from 1 day to 60 days, from 1 day to 45 days,from 1 day to 30 days or from 3 days to 21 days.

In all embodiments of the present invention the effective period of timeis preferably within a period of about 0.05 to about 72 hours,preferably about 1 to about 48 hours prior to the envisaged stresssituation such as in particular heat stress and/or transportation fromone location to another location; or within a period of about 0.05 toabout 72 hours, preferably about 1 to about 48 hours after the stresssituation such as in particular heat stress and/or transportation fromone location to another location; or within a period of stress such asin particular during heat stress and/or during transportation from onelocation to another location.

The term ‘an effective amount’ as used herein refers to an amountnecessary to obtain the desired reduction of the stress level. Thephysiological effect may be achieved by one single dose or by repeateddoses. The effective amount of the methane inhibitor in the methodsaccording to the invention may vary depending upon known factors, suchas the physiological characteristics of the particular composition andits mode and route of administration; the age, health and weight of theruminant; the nature and extent of the symptoms; the kind of concurrenttreatment; the frequency of treatment; and the effect desired which canbe determined by the expert in the field with normal trials, or with theusual considerations regarding the formulation.

In all embodiments of the present invention, the effective amount of themethane inhibitor to be administered to the ruminants is preferablyselected in the range of 0.05 to 5 methane inhibitor/animal/day, morepreferably in the range of 0.1 to 4 g methane inhibitor/animal/day, mostpreferably in the range of 0.5 to 3 g methane inhibitor/animal/day.

Advantageously, in all embodiments of the present invention, the methaneinhibitor respectively the composition comprising the methane inhibitoris administered prophylactically, or when the animal has or is at aparticular risk of developing stress such as preferably beforetransportation and/or slaughtering or if heat stress is to be foreseene.g. in particular hot summers.

Thus, in one preferred embodiment the invention relates to a method ofenhancing the endurance to stress in ruminants which is at risk ofdeveloping stress, preferably stress due to transportation and/orslaughtering, said method encompassing the administration of aneffective amount of a methane inhibitor respectively a compositioncomprising the methane inhibitor to the ruminant at least 1 day beforetransportation and/or slaughtering, preferably at least 5 days, morepreferably a least 10 days before transportation and/or slaughtering.Preferably, the methane inhibitor respectively a composition comprisingthe methane inhibitor is administered at least once a day starting 20days prior to the risk of developing stress, preferably 10 days prior tothe risk of developing stress, most preferably 5 days prior to the riskof developing stress. The method is in particular suitable for beefcattle and sheep.

In another preferred embodiment, the invention relates to a method ofreducing the severity of heat stress in ruminants, said methodencompassing the administration of an effective amount of a methaneinhibitor respectively a composition comprising the methane inhibitor toa ruminant exposed to or to be exposed to heat stress. Heat stress mayoccur when a ruminant is exposed to a humidity index of 68 or greater,75 or greater, or >79.

In all embodiments of the present invention, domestic cattle, sheep andgoat are the more preferred species. For the present purposes, mostpreferred species are domestic cattle. The term includes all races ofdomestic cattle, and all production kinds of cattle, in particular dairycows and beef cattle.

A particularly preferred group of ruminants in all embodiments accordingto the present invention are dairy cows, in particular lactating dairycows or beef cattle.

Dairy cows may be administered the methane inhibitor respectively thecomposition comprising the methane inhibitor before lactation, duringlactation or after lactation onset. If the composition is administeredbefore lactation, this can be from 90 days prior to lactation onset to 1day prior to lactation onset, preferably from 45 days prior to lactationonset to 10 days prior to lactation onset. The composition may beadministered daily prior to lactation onset.

The methane inhibitor respectively the composition comprising themethane inhibitor may also be administered pre- or post-calving for asuitable number of days. For example, the composition may beadministered to the animal for 40 days to 100 days post calving, or for45 days to 95 days post calving, or for 50 days to 90 days post calving.

The composition according to the present invention may be formulated inany suitable form, including a powder, a granule, a pellet, a solution,or a suspension.

In one embodiment, the composition can be a dry, free-flowing powder(powderous formulation) suitable for direct inclusion into acommercially-available feed or as a supplement to a total mixed rationor diet. The powderous formulation may be mixed with either solid orliquid feed or with water. In another embodiment, the composition can beformed into pellets.

In all embodiments of the present invention, the composition comprisingthe methane inhibitor preferably is a powderous formulation comprisingthe methane inhibitor and a carrier material. Suitable carrier includesany carrier well known in the food and feed industry such as siliconedioxide without being limited thereto.

If the composition is a powderous formulation comprising the methaneinhibitor and a carrier material, the methane inhibitor is usuallysprayed onto or admixed with the carrier material by standard methods inthe art, e.g. by using solvent suitable for the preparation of food orfeed products such as e.g. dichloromethane followed by evaporation ofthe organic solvent.

Alternatively, the methane inhibitor can be diluted in a suitable edibleoil before being sprayed onto or admixed with the carrier material. Thepowderous formulation may in addition contain usual additives used inthe preparation of powderous formulations for feed application.

The amount of the methane inhibitor in the composition according to thepresent invention, in particular in a powderous formulation ispreferably selected in the range of 1 to 20 wt.-%, preferably in therange of 2 to 15 wt.-%, most preferably in the range of 4 to 12 wt.-%,based on the total weight of the composition.

The methane inhibitor respectively the composition comprising themethane inhibitor according to the present invention such as inparticular the powderous formulation is preferably administered admixedwith the animal's feed, wherein the term feed refers to any compound,preparation, mixture, or composition suitable for, or intended forintake by an animal. Exemplary feed for ruminants such as cows includeforage (grass, legumes, silage), hay, grass, grain as well as soywithout being limited thereto.

When incorporated directly into the animal's feeds, the methaneinhibitor respectively the composition comprising the methane inhibitoraccording to the present invention may be added in amounts ranging from0.1 to 100 kg per ton, such as from 0.1 to 20 kg per ton (2000 pounds)of feed. In some embodiments, the composition can be added to animalfeedstuffs in amounts from 0.1 kg to 50 kg per ton, from 0.1 to 20 kgper ton, or from 0.5 kg to 10 kg per ton of feed. In certainembodiments, the composition may be added to feeds in amounts rangingfrom 1 to 5 kg per ton of feed.

When expressed as a percentage of dry matter of feed, the methaneinhibitor respectively the composition comprising the methane inhibitormay be added to animal feedstuffs in amounts ranging from 0.01 to 2.5%by weight, such as from 0.0125% to 2% by weight. In one embodiment, thecomposition can be added to animal feedstuffs in amounts from 0.05 to1.5% by weight, such as from 0.06% to 1% by weight. In anotherembodiment, the composition can be added in amounts from 0.1 to 0.7% byweight, such as from 0.125% to 0.5% by weight of feed.

Alternatively, the methane inhibitor respectively the compositioncomprising the methane inhibitor such as in particular the powderousformulation according to the present invention may be fed directly tothe animal as a supplement in amounts of from 0.01 gram to 20 gram perkilogram of live body weight per day, such as from 0.01 gram to 10 gramper kilogram, 0.01 gram to 5 gram, 0.01 gram to 1 gram, 0.015 gram to 1gram, or 0.02 gram to 0.4 gram per kilogram of live body weight per day.

One of skill in the art can appreciate that the amount of the methaneinhibitor respectively the composition comprising the methane inhibitorfed can vary depending upon the amount of methane inhibitor incorporatedin the composition, the animal species, the size of the animal and thetype of the feedstuff to which the claimed methane inhibitorrespectively the composition comprising the methane inhibitor is added.

In a particular advantageous embodiment, the methane inhibitorrespectively the composition comprising the methane inhibitor is usedfor treating animals that already suffer from stress induced by theirenvironment, heat, nutrition, and/or transportation, in particular byheat and transportation.

Preferably, in all embodiments of the present invention the endogenousmethylsulfonylmethane in the rumen fluid is increased by at least 100%,preferably by at least 200%, more preferably by at least 300% based onthe control by the administration of the methane inhibitor respectivelythe composition comprising the methane inhibitor.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXPERIMENTAL PART

Eight fistulated Brahman steers (Bos indicus) were randomly allocated totwo groups (4 animals per group) and received a forage ad libitum diet(Rhode grass hay (Chloris gayana), chemical composition: DM, 917 g/kgfresh matter; in g/kg of DM: OM, 806; CP, 169; NDF, 661; ADF, 359; ADL,46; ash, 116 and GE 17.38 MJ/kg). The treatments used were chloroformfixed in cyclodextrin (reference) and propanediol mononitrate (10%purity on silicon dioxide). Animals were adapted to the diet over a 21 dperiod. After that initial period, experimental animals were placed intoindividual pens in an animal house for the measurement of intakes (10 d)and were treated with cyclodextrin (2 g/100 kg LW) and molasses (60mL/d). On the last 2 days animals were placed into open-circuitrespiration chambers for collection of rumen samples. Following theinitial adaption/control period one group of animals received thechloroform+60 mL of molasses during 21 days (1.6 g choloroform−CD/100 kgLW) and the second group received the propanediol mononitrate treatmentduring 21 days (2.5 g propanediol mononitrate/animal/day). propanediolmononitrate was provided to the animals mixed with molasses (60 mL/day,molasses were previously diluted in water: 1:4 water:molasses) and mixedwith the hay at three different times: 0 h, 3 h and 5 h after the feedwas offered. propanediol mononitrate group was treated with comparativeamounts of cyclodextrin as the chloroform group during the experiment.On days 20 and 21 of treatment both groups were placed in open-circuitrespiration chambers for rumen fluid collection.

Rumen fluid samples (approx. 60 mL per animal) were collected using aprobe with 2 layers of cheesecloth through the cannula of the animal at3 h post feeding, during confinement in respiration chambers todetermine rumen metabolites. The metabolites were quantified usingNuclear Magnetic Resonance spectroscopy in the NMR facilities of theInstitute for Molecular Bioscience and of the Queensland NMR Network(QNN) at the University of Queensland in

TABLE 1 Effects of PDMN on rumen metabolites compared with controlperiod (μmol/L rumen fluid). Metabolite Control PDMN SEM^(a) P-valueDimethylsulfone 20 113 3.84 0.001 ^(a)SEM, standard error of the mean

TABLE 2 Effects of chloroform on rumen metabolites compared with controlperiod (μmol/L rumen fluid) Control Chloroform SEM^(a) P-valueDimethylsulfone 21 80 3.85 0.007 ^(a)SEM, standard error of the mean

As can be retrieved, the treatment with the methane inhibitorspropanediol mononitrate and chloroform lead to a statisticallysignificant increase of the methyl donor, while propanediol mononitrateis particularly effective.

The invention claimed is:
 1. A non-therapeutic method for alleviating orreducing stress symptoms of a ruminant, the method comprisingadministering to the ruminant in need of alleviation or reduction ofstress symptoms an effective amount of propanediol mononitrate orchloroform to increase endogeneous methyl donors in rumen fluid of theruminant prior to experiencing stress, while experiencing stress and/orafter having experienced stress.
 2. The method according to claim 1,wherein the propanediol mononitrate or chloroform is administered atleast 1 day prior to the ruminant experiencing stress.
 3. The methodaccording to claim 1, wherein the propanediol mononitrate or chloroformis administered at least 5 days prior to the ruminant experiencingstress.
 4. The method according to claim 1, wherein the propanediolmononitrate or chloroform is administered at least 10 days prior to theruminant experiencing stress.
 5. The method according to claim 1,wherein the amount of the propanediol mononitrate or chloroform issufficient to increase methylsulfonylmethane in rumen fluid of theruminant.
 6. The method according to claim 1, wherein the propanediolmononitrate or chloroform is administered in the form of a powderousformulation comprising the propanediol mononitrate or chloroform and acarrier material.
 7. The method according to claim 6, wherein thecarrier material is silicone dioxide.
 8. The method according to claim1, wherein the ruminant is selected from the group consisting ofdomestic cattle, sheep and goats.
 9. The method according to claim 8,wherein the domestic cattle is selected from the group consisting ofdairy cows and beef cattle.
 10. The method according to claim 1, whereinthe propanediol mononitrate or chloroform is administered to beef cattlebefore transportation and/or slaughtering.
 11. The method according toclaim 10, wherein the propanediol mononitrate or chloroform isadministered to the beef cattle at least 1 day prior to thetransportation and/or slaughtering of the beef cattle.
 12. The methodaccording to claim 10, wherein the propanediol mononitrate or chloroformis administered to the beef cattle at least 5 days prior to thetransportation and/or slaughtering of the beef cattle.
 13. The methodaccording to claim 10, wherein the propanediol mononitrate or chloroformis administered to the beef cattle at least 10 days prior to thetransportation and/or slaughtering of the beef cattle.